Construction of a Functional Nucleic Acid-Based Artificial Vesicle-Encapsulated Composite Nanoparticle and Its Application in Retinoblastoma-Targeted Theranostics.

Retinoblastoma (RB) is an aggressive tumor of the infant retina. However, the ineffective targeting of its theranostic agents results in poor imaging and therapeutic efficacy, which makes it difficult to identify and treat RB at an early stage. In order to improve the imaging and therapeutic efficacy, we constructed an RB-targeted artificial vesicle composite nanoparticle. In this study, the MnO2 nanosponge (hMNs) was used as the core to absorb two fluorophore-modified DNAzymes to form the Dual/hMNs nanoparticle; after loaded with the artificial vesicle derived from human red blood cells, the RB-targeted DNA aptamers were modified on the surface, thus forming the Apt-EG@Dual/hMNs complex nanoparticle. The DNA aptamer endows this nanoparticle to target the nucleolin-overexpressed RB cell membrane specifically and enters cells via endocytosis. The nanoparticle could release fluorophore-modified DNAzymes and supplies Mn2+ as a DNAzyme cofactor and a magnetic resonance imaging (MRI) agent. Subsequently, the DNAzymes can target two different mRNAs, thereby realizing fluorescence/MR bimodal imaging and dual-gene therapy. This study is expected to provide a reliable and valuable basis for ocular tumor theranostics.

Recent theranostic applications of hydrogen peroxide-responsive nanomaterials for multiple diseases.

It is well established that hydrogen peroxide (H2O2) is associated with the initiation and progression of many diseases. With the rapid development of nanotechnology, the diagnosis and treatment of those diseases could be realized through a variety of H2O2-responsive nanomaterials. In order to broaden the application prospects of H2O2-responsive nanomaterials and promote their development, understanding and summarizing the design and application fields of such materials has attracted much attention. This review provides a comprehensive summary of the types of H2O2-responsive nanomaterials including organic, inorganic and organic-inorganic hybrids in recent years, and focused on their specific design and applications. Based on the type of disease, such as tumors, bacteria, dental diseases, inflammation, cardiovascular diseases, bone injury and so on, key examples for above disease imaging diagnosis and therapy strategies are introduced. In addition, current challenges and the outlook of H2O2-responsive nanomaterials are also discussed. This review aims to stimulate the potential of H2O2-responsive nanomaterials and provide new application ideas for various functional nanomaterials related to H2O2.

Monitoring leaching of Cd2+ from cadmium-based quantum dots by an Cd aptamer fluorescence sensor.

Quantum Dots (QDs) have been demonstrated with outstanding optical properties and thus been widely used in many biological and biomedical studies. However, previous studies have shown that QDs can cause cell toxicity, mainly attributable to the leached Cd2+. Therefore, identifying the leaching kinetics is very important to understand QD biosafety and cytotoxicity. Toward this goal, instrumental analyses such as inductively coupled plasma mass spectrometry (ICP-MS) have been used, which are time-consuming, costly and do not provide real-time or spatial information. To overcome these limitations, we report herein a fast and cost-effective fluorescence sensor based a Cd2+-specific aptamer for real-time monitoring the rapid leaching kinetics of QDs in vitro and in living cells. The sensor shows high specificity towards Cd2+ and is able to measure the Cd2+ leached either from water-dispersed CdTe QDs or two-layered CdSe/CdS QDs. The sensor is then used to study the stability of these two types of QDs under conditions to mimic cellular pH and temperature and the results from the sensor are similar to those obtained from ICP-MS. Finally, the sensor is able to monitor the leaching of Cd2+ from QDs in HeLa cells. The fluorescence aptamer sensor described in this study may find many applications as a tool for understanding biosafety of numerous other Cd-based QDs, including leaching kinetics and toxicity mechanisms in living systems.

Aptamer-Based Probes for Cancer Diagnostics and Treatment.

Aptamers are single-stranded DNA or RNA oligomers that have the ability to generate unique and diverse tertiary structures that bind to cognate molecules with high specificity. In recent years, aptamer researches have witnessed a huge surge, owing to its unique properties, such as high specificity and binding affinity, low immunogenicity and toxicity, and simplicity of synthesis with negligible batch-to-batch variation. Aptamers may bind to targets, such as various cancer biomarkers, making them applicable for a wide range of cancer diagnosis and treatment. In cancer diagnostic applications, aptamers are used as molecular probes instead of antibodies. They have the potential to detect various cancer-associated biomarkers. For cancer therapeutic purposes, aptamers can serve as therapeutic or delivery agents. The chemical stabilization and modification strategies for aptamers may expand their serum half-life and shelf life. However, aptamer-based probes for cancer diagnosis and therapy still face several challenges for successful clinical translation. A deeper understanding of nucleic acid chemistry, tissue distribution, and pharmacokinetics is required in the development of aptamer-based probes. This review summarizes their application in cancer diagnostics and treatments based on different localization of target biomarkers, as well as current challenges and future prospects.

Recent progress in developing fluorescent probes for imaging cell metabolites.

Cellular metabolites play a crucial role in promoting and regulating cellular activities, but it has been difficult to monitor these cellular metabolites in living cells and in real time. Over the past decades, iterative development and improvements of fluorescent probes have been made, resulting in the effective monitoring of metabolites. In this review, we highlight recent progress in the use of fluorescent probes for tracking some key metabolites, such as adenosine triphosphate, cyclic adenosine monophosphate, cyclic guanosine 5'-monophosphate, Nicotinamide adenine dinucleotide (NADH), reactive oxygen species, sugar, carbon monoxide, and nitric oxide for both whole cell and subcellular imaging.

Folic acid-modified fluorescent dye-protein nanoparticles for the targeted tumor cell imaging.

Serum albumin has a wide range of applications in biochemical experiments and pharmaceutical field. We found that a cyanine dye, dimethylindole red (Dir), could selectively interact with bovine serum albumin (BSA). Dir exhibited very weak red fluorescence, while the fluorescence intensity at 630 nm was enhanced up to 130-fold upon noncovalently interacting with 30 µM BSA. Besides, Dir showed a highly selective response to BSA over human serum albumin (HSA). For the detection of BSA, a limit of detection as low as 23 nM was obtained. Then biocompatible Dir-BSA nanoparticles were prepared by the desolvation technique. The Dir-BSA nanoparticles possess excellent fluorescence properties with a quantum yield of 32%. Furthermore, folic acid as a targeting group was conjugated to Dir-BSA nanoparticles and these nanoparticles were characterized by TEM and laser particle analyzer, etc. Folic acid-modified Dir-BSA nanoparticles were successfully used for tumor cell-targeted imaging.

Aptamer-integrated α-Gal liposomes as bispecific agents to trigger immune response for killing tumor cells.

A novel bispecific α-Gal liposome was constructed by self-assembling AS1411 aptamers into the α-Gal containing liposomes. The α-Gal liposomes were prepared using cell membranes of red blood cells from rabbit, which are composed of cholesterol, phospholipids, and α-Gal glycolipids. AS1411 is a DNA aptamer with high specificity and affinity for nucleolin and could integrate into liposomes by the modification of cholesterol. The bispecific α-Gal liposomes surface-functionalized by α-Gal and AS1411 aptamer could recognize anti-Gal antibodies and nucleolin overexpressed by tumor cells simultaneously, followed by activating the immune system to attack the tumor cells, resulting in the lysis of the tumor cells by antibody dependent cell-mediated cytotoxicity. Under simulated tumor environment, the lysis rate of MCF-7 cells treated by the AS1411 modified α-Gal liposomes drastically increased compared to the liposomes without AS1411 aptamer. This study suggests that the AS1411 modified α-Gal liposomes can recognize nucleolin-overexpressing tumor cells selectively, subsequently improve the effect of the immunotherapy with high specificity. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1176-1183, 2019.

Self-Assembled saRNA Delivery System Based on Rolling Circle Transcription for Aptamer-Targeting Cancer Therapy.

With the capacity of gene promotion, RNA activation (RNAa) has been supposed to be a powerful technique in the field of biomedicine, especially in an antitumor aspect. However, one of the pressing challenges for clinical application is how to efficiently deliver therapeutic probes to cancer. Herein, we synthesized a carrier through rolling circle transcription (RCT) to deliver p21 saRNA with high loading rate and targeting capacity. This carrier could be condensed from a micron dimension to about 200 nm by polyethylenimine (PEI). In addition, the trait of robust tumor targeting and improved cytotoxicity was demonstrated when the aptamer was modified through layer-by-layer self-assembly. Moreover, the 4-fold activation of the p21 gene could be obviously detected in a targeting group. Meanwhile, the cell apoptosis induced by p21 promotion was also exhibited, which indicated that this highly efficient saRNA delivery carrier had a specific antitumor effect and could reduce side effects to normal cells. Therefore, this delivery system had the potential to be used in RNAa applications and cancer-targeted treatment.

Self-assembled RNAi nanoflowers via rolling circle transcription for aptamer-targeted siRNA delivery.

To deliver siRNA efficiently, prevailing conventional lipid or polymer encapsulation often needs multi-step compounding methods, which may inevitably introduce cationic or other components and may lead to cytotoxicity or an immune response. Herein, we present a novel enzymatic synthetic approach to produce tumor-targetable RNAi nanoflowers. The RNAi nanoflowers are mainly composed of multiple tandem copies of siRNA precursors by rolling circle transcription (RCT), and produce large amounts of siRNA to silence Bcl-2 gene expression after cellular uptake, which can overcome the problem of low loading capacity. In particular, the RNAi microspheres (RNAi-MS) were condensed into nanosized complexes (RNAi nanospheres, RNAi-NS) by cholesterol-modified DNA strands without the assistance of polycationic agents. RNAi-NS are entirely composed of nucleic acid, giving them lower cytotoxicity and immunogenicity, which can be caused by synthetic polycationic reagents. In addition, the RNAi nanoflowers can also integrate DNA aptamers that bind specifically to target membrane proteins for cell-targeting. Therefore, thousands of copies of siRNA will be delivered to cells specifically, and this RNAi nanoflower system will have great potential for siRNA delivery and biomedical applications.

In vitro selection of DNA aptamers against renal cell carcinoma using living cell-SELEX.

Renal cell carcinoma (RCC) is the most common form of kidney cancer with poor prognosis. Early diagnosis of RCC would significantly improve patient prognosis and quality of life. In this work, we developed new aptamer probes for RCC by using cell-SELEX (systematic evolution of ligands by exponential enrichment) only after 12 rounds of selection, in which a clear cell renal cell carcinoma (ccRCC) cell line 786-O was used as target cell, and embryonic kidney cell line 293T as negative control cell. The selected aptamers were subjected to flow cytometry and laser confocal fluorescence microscopy to evaluate their binding affinity and selectivity. The dissociation constant Kd values of four selected aptamers are all in the nanomolar range. Aptamer W786-1 with the best binding affinity and a Kd value of 9.4 ± 2.0nM was further optimized and its truncated sequence W786-1S showed considerable affinity to 786-O cells. The proteinase and temperature treatment experiment indicated that W786-1 could recognize the target 786-O cells through surface proteins, and remain good binding affinity and excellent selectivity under physiological conditions. Therefore, on the basis of its excellent targeting properties and functional versatility, W786-1 holds great potential to be used as a molecular probe for identifying and targeting RCC.

Gd2O3 and GH combined with red blood cells to improve the sensitivity of contrast agents for cancer targeting MR imaging.

Herein, we fabricated efficient MR imaging probes by incorporating gadolinium oxide nanoparticles (Gd2O3) and gadolinium hybrid nanoparticles (GH) within RBCs. The Gd2O3 and GH encapsulated in the RBCs exhibited high relaxation rates and revealed high sensitivity for T1 MR imaging.

Functional Hyperbranched Polylysine as Potential Contrast Agent Probes for Magnetic Resonance Imaging.

Researchers have never stopped questing contrast agents with high resolution and safety to overcome the drawbacks of small-molecule contrast agents in clinic. Herein, we reported the synthesis of gadolinium-based hyperbranched polylysine (HBPLL-DTPA-Gd), which was prepared by thermal polymerization of l-lysine via one-step polycondensation. After conjugating with folic acid, its potential application as MRI contrast agent was then evaluated. This contrast agent had no obvious cytotoxicity as verified by WST assay and H&E analysis. Compared to Gd(III)-diethylenetriaminepentaacetic acid (Gd-DTPA) (r1 = 4.3 mM(-1) s(-1)), the FA-HBPLL-DTPA-Gd exhibited much higher longitudinal relaxivity value (r1 = 13.44 mM(-1) s(-1)), up to 3 times higher than Gd-DTPA. The FA-HBPLL-DTPA-Gd showed significant signal intensity enhancement in the tumor region at various time points and provided a long time window for MR examination. The results illustrate that FA-HBPLL-DTPA-Gd will be a potential candidate for tumor-targeted MRI.

DNA sequence-dependent fluorescence of doxorubicin for turn-on detection of biothiols in human serum.

Doxorubicin (Dox) is a DNA-targeting anthracycline antibiotic active against a wide spectrum of cancers. The interaction between Dox and double-stranded DNA (dsDNA) was used to load Dox using DNA duplexes as carriers. More importantly, the interesting DNA sequence-dependent fluorescence response of Dox could be exploited in the design of efficient Dox release systems and efficient fluorescence sensors. In this work, we demonstrated that separate introduction of G and C bases into T-rich single-stranded DNA (ssDNA) sequences afforded the best discrimination of Dox binding between dsDNA and ssDNA. For the first time, we successfully utilized this interesting DNA sequence-dependent fluorescence response of Dox as a signal transduction mechanism for the sensitive detection of biothiols in human serum. Cysteine, homocysteine, and glutathione were detected at as low as 26 nM, 37 nM, and 29 nM, respectively. The biosensors exhibited not only good selectivity, stability, and sensitivity in aqueous solutions but also a sensitive response in human serum, demonstrating their potential for diagnosis.

A Cellular Compatible Chitosan Nanoparticle Surface for Isolation and In Situ Culture of Rare Number CTCs.

Circulating tumor cell (CTC) isolation has attracted a great deal of research interest in recent years. However, there are still some challenges, including purity as well as viability of the captured CTCs, resulting from nanoscale structures and inorganic nanomaterials. Here, a chitosan nanoparticle surface is first fabricated by electrospray to provide a cellular compatible interface. The "soft" substrate, further modified by polyethylene glycol (PEG) as an antifouling molecule and DNA aptamer as a specific capture molecule, has a hydrophilic nature and is capable of specific capture of viable rare CTCs from artificial white blood cell (WBC) samples. Furthermore, a subsequent in situ culture strategy based on the developed cellular compatible soft interface is introduced for further purification and proliferation of the captured rare number target cells. The WBCs are weeded out after 2 d, and after a 7 d proliferation nearly 200 MCF-7 cells are obtained from 7 target cells with more than 90% purity. This work provides a promising strategy for viable isolation and purification of rare CTCs and it has great potential for achieving clinical validity.